FI72977C - FOERFARANDE FOER FRAMSTAELLNING AV EN NY THERAPEUTIC ANVAENDBAR FOERENING, 9- / 2-HYDROXI-1- (HYDROXIMETYL) ETOXI / METYL GUANIN. - Google Patents

Description

1 72977

The invention relates to a process for the preparation of a novel therapeutically useful compound, 9 - [(2-hydroxy-1-) (hydroxymethyl) ethoxy / methyl] guanine. ) for the preparation of ethoxymethylfunguanine.

Nucleosides consist of an N-ribose or 2-deoxy-D-ribose sugar unit chemically linked to a purine-10 or pyrimidine base, which is adenine, cytosine, guanine, thyrine or uracil, via a ring nitrogen atom of the base.

Because nucleosides and nucleotides are part of nucleic acids naturally occurring in cells, it has recently been hypothesized that they and their relatives could have potency as chemotherapeutic agents. However, their potential practical value is often diminished because they are easily deaminated by in vivo deaminases. Studies have been performed to determine the relationship between structure and activity for both adenosine deaminase substrates and inhibitors, some of which relate to nucleoside-like open-chain compounds. To date, however, despite numerous promising reports of novel compounds, no such compound suitable for chemotherapeutic use has been prepared, possibly with the exception of acycloguanosine, which is described in U.S. Patent 4,146,715 to Schaeffer.

It should be noted that the structure and groups of the compound of the invention are closely analogous to naturally occurring nucleosides and nucleotides. It has 30 necessary chain element order and length. The relevant O and OH functional groups, which actively bind to the biological centers in the biological environment, are in their natural positions and positions relative to the base, but possibly provided with protecting groups. In fact, the groups adjacent to the base are so similar in chemical composition to the deoxyribose compounds, 2,72977, that they may have the characteristic confirmation of a deoxyribose ring under suitable conditions. The basic difference is that the compound of the invention does not have the structural rigidity of a carbohydrate ring, which is why it differs unpredictably in its properties and behavior. In addition, the C-4 'position of the compound is not chiral, so no stereoisomers are present. Each hydroxyl group is primary. Glyoxide bond syn-anti-isomerism cannot occur.

The process for the preparation of a novel therapeutically useful compound G *, i.e. 9- (γ2-hydroxy-1- (hydroxymethyl) ethoxy] methyl] guanidine, is characterized by coupling to a purine base compound which is optionally chemically protected from its other active sites but which has a free> NH or = NM group in which M is chlorine, bromine or iodine, substituted silyl, in particular trimethylsilyl, lower alkyl or a mercury, silver or tin salt, a compound of the formula R.0- CH 0 -CH-CH 2 -R 0 2 O 12.22

O

! CH 2 Hal wherein R 1 and R 2 are independently hydrogen, benzyl or tert-butyldimethylsilyl, under suitable reaction conditions, and if R 1 and / or R 2 is other than hydrogen, treat the resulting compound to replace R 1 and R 2. with hydrogen.

Thus, a compound of the invention may be prepared by treating a suitably halogenated base with a suitable alkyl residue. The synthesis can be initiated by treating 1,3-dichloro-2-propanol with sodium benzylate 11a as a nitrogen shielding gas, followed by trioxymethylene and HCl to prepare the chloromethoxy derivative, taking care to remove excess water. From this 1,3-dibenzyloxy-2-propanol derivative, a suitably halogenated base such as 6-chloropurine can be prepared in DMF using triethylamine as an acid scavenger. Treatment of the chlorine compound thus formed with methanolic ammonia in a steel reaction vessel in the steel reaction vessel gives a 6-amino derivative. The product can be debenzylated to give a compound of the invention, for example by treatment with hydrogen in the presence of palladium oxide in methanol. The protecting groups are attached, if desired, by conventional known methods.

Alternatively, halogenated alkyl residues may be attached to a halogenated or non-halogenated purine base.

The compound of the invention has a very strong antiviral activity and is effective against, for example, Herpesvirus, vesicular stomatitis VSV virus and Varvicella zoster virus.

The compound of the invention may be administered to a patient parenterally, interstitially, topically as an ointment, spray or powder, as well as as eye or nasal drops or orally. In general, the modes of administration and dosage forms will follow known, published methods used for known antiviral drugs, such as acycloguanosine. An effective single dose when administering the compounds interstitially or parenterally is about 0.1 to 100 mg per kilogram of mammalian body weight as the free base, preferably 0.2 to 20 mg / kg, and most preferably about 5 mg / kg when administered 2-4 times daily.

Formulations for oral administration are preferably in the form of a fine powder or granules combined with diluents and / or dispersants and / or surfactants, for example, aqueous or syrupy dispersions or tablets or capsules. Solutions of the compounds in distilled water or physiological saline, for example isotonic, optionally phosphate buffered solutions, in a concentration of 1-20%, preferably 2-15% and most preferably about 10%, are suitable for parenteral or interectal administration. Ointments containing the compounds, for example those formulated in an oil-in-water ointment base, in a concentration of about 0.1-10%, preferably up to 3% and most preferably about 1% (w / v) of the active ingredient can be used to treat external infections.

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They can also be combined with paraffin oil or petrolatum as an emulsion, optionally with a stabilizing surfactant, or with dimethyl sulfoxide.

Investigation and evaluation of the anti-herpes virus effect

Herpes simplex virus (HSV) strains were grown and counted at 36 ° C in connective tissue mast cells from human fetal tissues used for virus growth.

Cells were grown in Eagle's medium (BME; Auto-Pow, 10 Flow Laboratories) supplemented with 0.112% sodium bicarbonate, 2 mM 1-glutanin, 3 mg neomycin per 100 rftl, and 5-20% calf serum. 5% BME, as described above, means a medium containing 5 ml of calf serum with a total volume of 100 ml.

The HSV titer is determined by the plaque titration method (Roizman & Roane, "Virology" 15 (1961) 75-79). Tissue culture dishes are inoculated with cells and used for experiments when approximately 75% of the surface is covered. Equal volumes (0.2 ml) of log-diluted virus solutions are inoculated into each tissue culture dish, adsorbed with stirring for 1 h, the inoculum is removed and 2 ml of 5% BME supplemented with 0.5% human serum immunoglobulin is added. After 48 hours of incubation (36 ° C, 5% C (>>)), the supernatant is removed and the cells are stained with 0.05% aqueous crystal violet, the number of plaques is counted, the replicates are averaged and the number of plaque-forming units (pfu) is counted.

The anti-herpes simplex activity of the compounds is studied using a freshly prepared thief-toluene solution of the compound prepared by dissolving 1.2 mg of the compound in BMEre. The compound is appropriately diluted in 5% BMEre containing 0.5% human serum immunoglobulin just prior to use.

Tissue culture dishes (35 x 10 mm) with about 35 75% cell layers are inoculated with a solution containing about 50 plaque-forming HVS units / 0.2 ml, and 5,72977 viruses are adsorbed with stirring for 1 h. After removal of the inoculum, 2 ml of 5% BME with 0.05% immunoglobulin and drug are added in triplicate. No drug is added to one plate group and is used as a 5 control group. After 48 hours of incubation (36 ° C, 5% CO 2), the supernatant is removed, the cells are stained as mentioned and the plaques are counted. The results from the different plates are averaged and the plaques present in each drug dilution are counted. The reduction in plaque size caused by each drug concentration compared to control samples is assessed visually. The reduction in the number of plaques indicates that the added compound inhibits the proliferation of viral cells. The decrease in the area of growing plaques indicates inhibition of plaque growth, i. inhibition of viral proliferation-15 caused by the drug.

The results showed that the compound G * of the invention was very effective against herpes simplex. The compound reduced plaque size by 25% even at concentrations as low as 0.02 μg / ml, 50 S at 0.1 pg / ml, and 75% at 0.8 pg / ml. The compound reduced the number of plaques by 25% at 0.04 pg / ml, 50% at 0.1 μg / ml and 75% at 0.2 μg / ml. At the highest concentration tested (250 ug / ml), it showed no signs of cellular toxicity. When the compound was used at a concentration of 2 μg / ml or more, there was no increase or decrease in plaques. The clear effect was noticeable even at a concentration as low as 0.007 μg / ml.

The compound of the invention was tested by conventional plaque titration methods to investigate its activity against several other viruses.

Experiments investigating the antiviral effect were performed with the plaque experiments described above. Compound G * was anti-VSV (vesicular stomatitis) virus at low concentrations, and coxsackievirus CVB3 at low concentrations.

Compound G * was also tested for Varicella Zoster virus, which causes chickenpox and shingles, by similar methods. It was found to be active against Varicella Zoster even at concentrations as low as 13 ug / ml.

Substantially similar results were obtained with 5 studies of G * 8 for a different HSV, type I strain, and 6 different HSV, type II strains.

Comparing the compound of the invention to the preferred compound of G * FI Patent 60,709, known as acyclovir, 9- (2-hydroxyethoxy-10-methyl) guanine, it appears that structural differences lead to greater utility of the compound of the invention. The activity of the compound G * according to the invention is at least three times that of the acyclovir. (EDj.q = concentration required to inhibit plaque formation by 50%). Compound Test Virus Virus ED ^ q G * HSV-1 0.09 acyclovir HSV-1 0.7

The invention is further illustrated by the following examples.

Example 1 Preparation of 2-N-acetyl-9-N, N-benzyloxy-1 - (benzyloxymethyl) ethoxymethylguanine (starting material) 2-N-acetylguanine (1.93 g, 10 mmol) and ammonium sulfate ( 100 mg) was suspended in 1,1,1,3,3,3-hexamethyldi-25 silazane (HMDS) (20 ml). The mixture was refluxed with stirring for 3 hours at which time it became clear. Excess HMDS was removed under reduced pressure with a hot water bath to give a white solid, silyl-protected 2-N-acetylguanine, which was used without further purification. The white solid was dissolved in DCE (50 mL) and 1,3-dibenzyloxy-2-chloromethoxypropane (5 mmol) was added followed by freshly distilled anhydrous stannous chloride (1 mL). The solution was allowed to stand overnight at room temperature. The mixture was poured into a mixture of aqueous sodium bicarbonate and chloroform and shaken. The mixture was filtered through celite to remove the precipitate. The phases were separated and the aqueous phase was extracted once with chloroform.

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The combined organic phases were washed with water, dried over anhydrous sodium sulfate and evaporated under reduced pressure to give 2.19 g of product. The reaction product was dissolved in chloroform (5 mL) and applied to a TLC silica gel-5 column (9 x 6.5 cm). Eluted first with chloroform (60 ml) and then with 2% methanol in chloroform. Three relatively pure fractions were obtained from tubes 15 (0.047 g), 19-20 (0.148 g) and 23-27 (0.43 g). Each of these fractions was crystallized from ethanol to give about 20 mg, 64 mg and 10,250 mg of product. The last fraction (250 mg) was determined by UV spectrum to be 2-N-acetyl-9-, β-benzyloxy-1- (benzyloxymethyl) ethoxy / methyl} guanine.

The product was recrystallized from ethanol and m.p. was 142-144 ° C. UV spectrum:> ·. (H00) 259, 278 (shoulder),

ItlclX Δ 15 (pH 1) 262, (pH 13) 263.

The compound is hereinafter referred to as N-acetyl-benzyl G * and has the structural formula:

I I

ch3-co-hn i CH_ i 2

25 O

^ O ^^ CH2-0-CH2-CH2-0-CH2 ^ Oy

Example 2 Preparation of 3 0 9- [2-hydroxy-1- (hydroxymethyl) ethoxy / methylarguanine

N-Acetylbenzyl G * (1.288 g, 0.00270 mL) prepared according to Example 1 was dissolved in pyridine (1.5 mL) and concentrated ammonium hydroxide (6 mL) was added to the mixture.

The vessel was tightly closed and placed in a water bath adjusted to 55 ° C. After 15 hours there was a form __________ -. ---- T7 ^ 8 72977 precipitated crystals which were filtered and washed with ethanol. Kdeiden sp. 170-177 ° C and recrystallized from ethanes (60 ml) to give 1.863 g (0.00191 mol, 70.7%) of 9 - [(2-benzyloxy-1-benzyloxyethyl) ethoxy] methyl] -5-guanine, mp. 180-182 ° C.

The compound prepared as above (0.665 g, 0.00139 mol) was dissolved in boiling ethanol (40 ml). Palladium oxide (0.67 g) was added to the mixture, followed by cyclohexene (20 ml). Reflux was continued and after 2 to 10 hours the product still contained a lot of starting material by TLC. Therefore, more palladium oxide (0.6 g, Aldrich Gold Label) was added. After 5.5 hours the reaction seemed to be still slow and therefore palladium black (0.5 g which had been stored under water for several months and now dried by filtration and washing with ethanol) was added, after 7 hours cyclohexene (15 ml) was added. After 12 hours the reaction was incomplete according to the TLC plate, but after 22.5 hours the reaction was complete. The reaction mixture was filtered hot and the catalyst was washed with hot 95% ethanol.

On cooling, crystals separated which were filtered and washed with 95% ethanol. 127 mg of crystals were obtained which did not melt below 360 ° C, although they turned dark brown. The catalyst still contained absorbed product, so it was washed with hot 75% ethanol. The wash-25 solution was combined with the above mother liquor and evaporated under reduced pressure. The residue was dissolved in a hot mixture of water (2.5 ml) and ethanol (2.5 ml) and then more ethanol (17.5 ml) was added with heating. The solution was allowed to crystallize. The crystals (155 mg) were filtered and washed with ethanol. Crystals 30 did not melt when heated to 360 ° C. About 60 mg of residue was obtained from the mother liquor. Thus, the product yield was 282 mg (0.00110 mol, 79%). UV spectrum: ν (EtOH) 254, 270 (shoulder), (H 2 O) 252, 269 (shoulder), (pH 1) 254, 272 (shoulder), (pH 13) 262.

The product is 9 - [[2-hydroxy-1- (hydroxymethyl) ethoxy] methyl] guanine, compound G *, having the structural formula: 9 o 72977 !! HN ^ \ - Ή v [ι> b CH_ I 2 0

HO-CH2-CH2-OH

Claims (2)

A process for the preparation of a new therapeutically useful compound, 9- [2-hydroxy-1- (hydroxymethyl) ethoxy] methyl (-5-guanine), characterized in that it is incorporated into a purine base compound which is optionally chemically protected from its other active sites but which is free of> NH- or = NM- group in which M is chlorine, bromine or iodine, substituted silyl, in particular trimethylsilyl, lower alkyl or a mercury, silver or tin salt, in the 9-position a compound of formula r10-ch2-ch-ch2 -or2 0 1. n CH2Hal wherein R1 and R2 are independently hydrogen, benzyl or tert-butyldimethylsilyl, under suitable reaction conditions, and if R1 and / or R2 is other than hydrogen, treating the resulting compound to replace R1 and R2 with hydrogen.